The invention relates to a device for imaging at least one microscopic property of a sample. Such devices are in particular used in the field of microscopy, in particular for analyzing biological or medical samples. Also other fields of use are possible. Further, the invention relates to the use of the device for a coherent laser spectroscopy method as well as to a method for imaging at least one microscopic property of a sample.
Particularly in the field of biology and medicine highly improved optical imaging techniques have been developed. In addition to the classical light microscopy that relies for the imaging on dispersion, reflection or absorption properties of a sample, increasingly methods are used where the molecular or atomic properties of the sample to be examined are used, or wherein the sample is modified in a predetermined manner (for example by dying or using marker molecules or marker groups), for improving by means of these modifications, the resolution and contrast of the image. Such methods are often referred to as “biophotonics”. An overview over such methods can for instance be gathered from S. Liedtke and J. Popp: “Laser, Licht and Leben”, Wiley-VCH Verlag GmbH & Co.KG aA, Weinheim, 2006, pages 111-133.
For the purpose of imaging, frequently the technology of fluorescence microscopy is used. For this purpose, a sample is illuminated by illumination light of one or more wavelengths and the light emitted by the sample as a reaction of this illumination light is a luminescent light of shifted frequency (i.e. of the fluorescence light and/or phosphorescence light) that is detected. For enhancing the spectral properties of the sample, frequently dying technology is used. For being able to also excite multiphoton transitions, frequently in addition to conventional light sources also lasers are used for exciting.
One difficulty mainly occurring in the course of fluorescence microscopy is to separate for the analysis the emitted luminescent light and the illumination and detection light from each other. For conventional fluorescence microscopes dichroic filters or mirrors are typically used that separate the illumination light from the luminescent light to be detected. Due to the Stokes shift, the luminescent light comprises typically a longer wavelength compared to the illumination light.
Such technology is, however, subject to various technical challenges. Such optical component parts with very specific spectral transparency or reflectivity properties are technically difficult to manufacture. Further, such component parts are typically non-flexible with regard to the transmitted or reflected wavelength so that a change of the illumination- and/or luminescence wavelength often requires a complete replacement of these selective optical elements. Another difficulty is that in many cases the illumination- and/or the luminescence-wavelength have a limited spectral width so that the illumination and luminescence light overlap spectrally. A separation of the illumination light and luminescence light only based on the wavelength is therefore very difficult.
From DE 198 42 153 C2 and DE 102 57 237 A1 technology is known making use of spatial separations of the illumination and luminescent light instead of a spectral separation. For the purpose of separation, the DE 198 42 153 C2 therefore suggest a partial transparent mirror that deflects light from the illumination light source to the object, but allows fluorescence light coming from the object to pass partially to the detector. For this purpose, the surface of the mirror comprises a reflecting mirror surface and a transparent mirror surface. The fluorescent light detected in the reverse direction illuminates the entire pupil plane and is therefore substantially in its entirety passing through the transparent areas of the spatial beam splitter.
Similarly, DE 102 57 237 A1 suggests means for a spatial separation of the illumination light from the detection light. Again, the arrangement shown comprises at least one reflecting first area and at least a transparent second area. The spatially splitting beam splitter is in this case positioned preferably in or close to the pupil plane. The fluorescent light detected in the reverse direction does also in this case illuminate the entire pupil plane and is therefore almost entirely passing the transparent areas of the spatially splitting beam splitter.
One advantage in using these spatially splitting beam splitters is that it is possible to abstain from using the spectral beam splitters that are disadvantageous and described above, as for example dichroic mirrors and beam splitters in combination with filter elements.
A disadvantage of the known fluorescence microscopes is that regardless of the described enhancements by using spatially splitting beam splitters the contrast has its limits, in particular in case of non-dyed or non-modified biological samples, as for instance in the case of CARS-microscopy. This results in particular from the detection irradiation that is in this case a coherent detection irradiation (also an irradiation of synchronously oscillating wave trains), is superseded by a background irradiation resulting in the background noise. Therefore, the signal-to-noise-ratio determines the contrast that can be achieved for the image in the case of the described fluorescence microscopy methods.
In addition to the described fluorescence methods a number of additional spectroscopic methods exist that can also be applied successfully in the field of biophotonics or microscopy. Among these methods in particular methods are mentioned that are based on the known principles of laser spectroscopy. One example for such methods is the so-called CARS-spectroscopy (coherent anti-Stokes Raman scattering) that is for example described as well in the above-mentioned publication by S. Liedtke and J. Popp. This spectroscopy is based on the simultaneous irradiation by photons of different excitation frequencies onto the sample wherein the difference in these excitation frequencies equals just to a frequency of a Raman-active oscillation transition in the sample. In this case the sample emits light of a frequency that equals the sum of an excitation frequency and the frequency of the Raman-active oscillation transition.
Also in case of CARS the problem occurs that the detection light comprises several portions of which one or more frequencies have to be detected separately and selectively. In particular the CARS-portion of the illumination light has to be detected separately from the Raman-irradiation. For the purpose of this separation several measuring schemes have been suggested, wherein the coherent properties of the detection light is used. One example for such a detection scheme is the so-called “gated heterodyne CARS”-method (GH-CARS), for example described in “High-contrast chemical imaging with gated heterodyne coherent anti-Stokes Raman scattering microscopy”, Appl. Physics B 81, 875 (2005).
These known methods for separating the coherent illumination light from the background irradiation are, however, technically very sophisticated, since, as for example in case of GH-CARS, time resolution electronics with complex measuring schemes has to be used. The complexity lowers the attractivity of such methods for use in low cost microscopy methods. Moreover, these methods are prone to failures.